SUIT-003 O2 ce D050

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SUIT-003 O2 ce D050

Description

Ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png

Abbreviation: CCP-ce Snv

Reference: A: - Coupling control protocol for living cells with plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv) - SUIT-003

SUIT number: D050_ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama

O2k-Application: O2

MitoPedia: SUIT - Plasma membrane-permeable succinate (NV118) in ROUTINE respiration. Coupling control protocol (CCP) in living cells (ce)
SUIT protocol pattern: ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama

SUIT-003 O2 ce D050 protocol is designed to study CI-deficiency in living cells. Plasma membrane-permeable succinate (MitoKit-CII/Succinate-nv, Snv), which is added in ROUTINE stimulates mitochondrial respiration in CI-deficient human blood cells, fibroblasts and heart fibres, acting on Complex II of the electron transfer system. Moreover, the SUIT-003 O2 ce D050 protocol allows to evaluate the coupling control of living cells. Respiratory capacities are tested in a sequence of coupling states: ROUTINE and ET. Optionally, to study LEAK-respiration, the phosphorylation system is inhibited by oligomycin. The final concentration of oligomycin has to be carefully optimized for various cell types, to minimize the inhibitory effect on the electron transfer system which would lead to an underestimation of ET-capacity.

Communicated by Doerrier C (last update 2019-09-24)

Representative traces

D050 O2 traces.png

MitoPedia: SUIT

Steps and respiratory states

Ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)


ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
ce1Snv ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
  • MitoKit-CII/Succinate-nv (diacetoxymethyl succinate) is a plasma membrane-permeable prodrug (permeable succinate; Snv) that diffuses across the plasma membrane. Cleavage of diacetoxymethyl residues is mediated by intracellular esterases, thus releasing succinate in the intracellular space. NeuroVive #: NV118
(ce2Omy) L(Omy) ce1;ce1Snv;(ce2Omy)
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
  • The addition of oligomycin is optional depending on the experimental question to resolve.
ce3U E ce1;ce1Snv;(ce2Omy);ce3U
ce4Rot ROX ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot
  • Rotenone inhibits Complex I, thus inhibiting the TCA cycle in living cells with intact plasma membrane. In the presence of Succinate-nv (diacetoxymethyl succinate, MitoKit-CII), the intracellular release of succinate independent of the TCA cycle leads to stimulation of respiration.
ce5Ama ROX ce1;ce1Snv;(ce2Omy);ce3U;ce4Rot;ce5Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated either after inhibition of CIII (e.g. antimycin A, myxothiazol), CIV (e.g. Cyanide) or in the absence of endogenous fuel-substrates. Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration.


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Strengths and limitations

+ The use of (MitoKit-CII/Succinate-nv allows studying succinate-linked respiration, or to overcome a CI-deficiency in living cells.
+ Reasonable duration of the experiment.
+ This is a cell coupling control protocol that provides information on ROUTINE and ET in the presence of (MitoKit-CII/Succinate-nv.
- This protocol is provided without oligomycin addition. To also include LEAK in the protocol, an event for the addition of oligomycin and a related mark can be added by editing the DLP file, creating a DLPU. However, the addition of oligomycin has to be carefully tested to avoid inhibitory effects in the evaluation of the ET-capacity.
- Careful washing is required after the experiment to avoid carry-over of inhibitors and uncoupler.

Compare SUIT protocols

  • SUIT-003 O2 ce D062 carrier control SUIT protocol for SUIT-003 O2 ce D050 in living cells. The carrier of the plasma membrane-permeable succinate (DMSO) is added in ROUTINE.
  • SUIT-003 O2 ce D060 CCP with cell permeable compounds (MitoKit-CII/Succinate-nv and MitoKit-CII/Malonate-nv).

References

MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry